Recently, microfluidic devices capable of conducting chemical reactions and assays on a single microchip have been developed. However, the method of detection has been mostly limited to laser-induced fluorescence (LIF) because of its simplicity and sensitivity. One of the limitations of LIF is that it requires the analyte of interest to be fluorescent. Since most compounds are not natural fluorophores, LIF is not an ideal detection method. Thus, in order to use LIF detection method, a derivatization step is often required to make compounds of interest amenable to LIF detection.
Mass spectrometry (MS) is currently being investigated as an alternative detection method for microfluidic devices. In this regard, electrospray ionization mass spectrometry (ESI-MS) is particularly suited due to the similarity in flow rates generated by the microchip (i.e., microfluidic device) with those required for ESI-MS. ESI-MS is a powerful tool that has been broadly applied to the structural analysis of biological molecules. In particular, it provides a facile means to interface liquid chromatographic (LC) systems and mass spectrometry (MS), creating a system that integrates separation with structural analysis and molecular identification. The development of LC-MS has revolutionized analytical chemistry and biochemistry.
In the post-genomic era, attention has turned from DNA sequencing to the more complex problem of analyzing how this genetic information directs cell function. The analysis of protein structure and function is one of the keys to this question. In particular, analysis methods currently under development are typically focused on identifying unknown proteins whose presence can be correlated with a function, disease state or reaction to potential drug candidates.
Mass spectrometry is a highly sensitive tool for the analysis of proteins. It enables the masses of fragment ions of proteins or peptides to be determined with high accuracy and with high sensitivity. High mass accuracy enables an accurate and specific sequencing of peptides. In combination with progress in genomic sequencing and bioinformatics, this enables the identification and characterization of unknown components of cells. In tandem with multidimensional gel electrophoresis methods, it provides a means to identify the complement of the proteins expressed by a cell under a defined set of conditions. This totality of expressed proteins is defined as the proteome.
Mass spectrometry is also developing from this simple “mining tool” for providing protein sequence information into more deeply integrated areas, such as functional characterization of biologically important genes, functional proteomics, quantitative mapping of cellular proteins and deciphering protein interaction networks. In addition to sequencing, mass spectrometry is currently the only tool available that can readily detect post-translational modifications (changes to protein structure after synthesis), such as phosphorylation and dephosphorylation and the actions of proteases that each plays critical roles in the control of cellular activity.
Another important MS application is the identification of molecules participating in the formation of macromolecular complexes. The study of molecular interactions is a rapidly developing field. The analysis of protein expression in cells (also known as proteomics) is therefore important in target identification and validation, and in ADME/PK (absorption-distribution-metabolism-excretion/pharmacokinetic) studies. However, such proteomic studies, in which proteins are identified by analysis of enzymatically produced peptide fragments, are expensive and labor-intensive. Technical difficulties exist in both sample separation and sample delivery systems for using ESI-MS in analysis of proteins, primarily because the samples that can be isolated from traditional gel-based electrophoresis are in very limited amounts. This makes them difficult to analyze in a traditional ESI-MS configuration.
To overcome some of the problems created by small sample sizes, interfaces capable of delivering low nanoliter per minute volumes of sample (so-called ‘nanospray’) to MS have been developed. These extend the time over which a very small amount of sample (e.g., 1 μL or less) can be delivered to the mass spectrometer, providing improved signal/noise ratios and thus sensitivity. However, Nano-ESI-MS is labor-intensive and slow (in current designs, sample loading and set-up of the electrospray capillary are both manual processes). In addition, it cannot be readily adapted to on-line capillary separation methods such as liquid chromatography or capillary electrophoresis. For these reasons, nanospray is most often used as a “static” or off-line method in which samples are analyzed one-at-a-time, representing a serious bottleneck in applications that requires high throughput. Software that integrates the variety of analytical methods required to perform high throughput analysis using these systems is already available, thus design of a robust multi-use interface is the bottleneck in adapting nanospray to high throughput applications.
Microfluidic device based electrospray sources for use in mass spectrometry have recently been developed; see for example, Oleschuk and Harrison, Trends in Anal. Chem., 2000, 19, 379-388, and Licklider et al., Anal. Chem., 2000, 72, 367-375. However, these methods utilize non-elastic microfluidic devices and require fabricating an electrospray nozzle directly on the microfluidic device or attaching a capillary electrospray emitter to the microfluidic device. Unfortunately, fabrication of an electrospray nozzle directly on the microfluidic device increases the manufacturing complexity, the production time and the cost. Methods for attaching a capillary electrospray emitter to current microfluidic devices also have severe limitations. For example, the junction between the microfluidic device and the electrospray nozzle emitter requires a tight seal to avoid fluid sample leakage. More significantly, it is difficult to attach an electrospray emitter to non-elastic microfluidic device without introducing a certain amount of void volume. Furthermore, the electrospray emitter must be carefully attached to the microfluidic device making mass production using batch processes difficult.
Moreover, in these microfluidic devices the flow of fluid is typically electroosmotically driven or by applying pressure directly on the inlet portion of the microfluidic devices. These fluid flow methods further limit the utility of these microchips. For example, use of electroosmotic flow is incompatibility with many buffer systems, may cause molecular dissociation, and molecules can be damaged or degraded due to exposure to electric fields. Most importantly the ionic buffers required to drive electroosmotic flow interfere with electrospray ionization and limit its usefulness. The use of electric fields is also incompatible applications that demand the use of non-aqueous solvents.
Therefore, there is a need for a microfluidic device which comprises a means for providing a sample of fluid to an analytical device which does not require fabrication of sample providing means directly on the microfluidic device??. There is also a need for a microfluidic device in which a readily available electrospray emitter can be easily attached. There is also a need for a microfluidic device which does not require electroosmotic flow or electrophoresis or a direct application of pressure on the inlet portion of the microfluidic device.